We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).
Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.
Telomere protects chromosomal ends from aberrant fusion and degradation. Mammalian telomere is composed of G-rich hexameric repeats, TTAGGG, and shelterin complexes. The telomere repeat sequence of the nematode is similar to those of mammals (TTAGGC). Most eukaryotes utilize telomerase to add telomere repeats to their chromosomal ends. However, 10 – 15% of cancer cells utilize telomerase independent mechanism, known as Alternative Lengthening of Telomeres (ALT)3. Previously, we reported that telomere repeats and its associated sequences, named as TALT, were amplified in the telomeres of telomerase mutant lines that survived critical sterility2.
Telomere length was measured by quantitative PCR or by Southern blot, which provides average length of total telomeres4,5,6,7. Read count of telomere repeat in whole genome sequencing data is also an indicator of total telomere contents8. Although Single TElomere Length Analysis (STELA) could provide the length of a single telomere, it cannot provide spatial information of telomeres9. While POT-1::mCherry reporter protein provides the spatial information of telomeres in vivo, it cannot represent lengths of double-stranded telomeres, as POT-1 is a single-strand telomere binding protein10.
While aforementioned methods provide the averaged information of repetitive sequences, fluorescence in situ hybridization (FISH) allows to observe the amount and spatial pattern of individual sequences of interest on a chromosomal scale. Instead of purification of DNA, tissues or cells are fixed to preserve the native spatial information in FISH. Thus, FISH is a both quantitative and qualitative tool for observation of individual repeat sequences, such as telomere repeats.
This protocol provides an efficient method for simultaneous detection of both telomere and other repeats based on improvements from previously described methods 11,12. C. elegans larvae or adults are multicellular organism with highly differentiated cells. The heterogeneity of cells impedes on the quantitative analysis of a large number of telomere spots. To maximize the number of cells analyzed, embryos are isolated and spread on the polylysine-coated slides for FISH. In addition, this protocol can also be combined with immunofluorescence.
As a proof that the protocol works, we show that it is possible to observe and quantify two different repetitive sequences. DNA probe against TALT1 was generated with simple PCR incorporating digoxigenin-dUTP. Then this TALT1 probe and fluorescence-labeled telomere PNA probe were hybridized simultaneously. Subsequently, digoxigenin was detected by canonical immunofluorescence methods. We present here the representative images where TALT1 colocalized with the telomere in trt-1 survivors.
1. Labeling Probes with Digoxigenin-dUTP by PCR
2. Preparing Polylysine Coated Slides
Note: The entire procedure takes about 2 hr. Most of the steps are done at room temperature except for the drying step.
3. Fixation of Worms on the Slide Glass (Figure 1)
4. Fixation and Permeabilization
5. Hybridization of Fixed Cells
6. Washes and Immunofluorescence
7. Mounting and Observation
8. Quantification of Telomere Signal
Note: Quantification was done as described previously16. All the images that are to be compared should be taken with same setting including exposure time and light source.
It was previously reported that ALT survivor can emerge from telomerase-deficient mutant, trt-1(ok410), in low frequency by replicating internally localized 'Template of ALT' (TALT) sequences for telomere maintenance2. Using PNA probe, we were able to visualize telomeres in the dissected gonads (Figure 2A). The faint telomere signal was detected both in trt-1(ok410) and ALT survivor. The fuzzy signal was overlapped only with DAPI, suggesting that they may not be autofluorescence. Interstitial telomere-like repeat (ITR) is consistently observed in TRF assay in the study of C. elegans telomere4,10. Considering high specificity of PNA probe, they are likely to be the ITR dispersed throughout the genome.
The number of telomere spots was approximately 9 per pachytene nucleus in trt-1 (ok410). In the previous study, 12 foci was observed by POT-1::mCherry protein, which binds to single stranded telomere DNA10. Maximum of 24 foci per nucleus was observed in the wild type embryos17. The result suggests that mCherry reporter method is better for the experiment where the number of telomeres should be counted. However PNA FISH is able to detect double-stranded telomere DNA as well as single-stranded telomere DNA in proportion with the telomere length. In contrast, the number of telomere spots was approximately 7 in ALT survivor, which have fused chromosomes (N = 3) 2. This result is consistent with the prediction that telomere spots would be 6 in ALT survivor. We concluded that the signal intensity of ALT survivor was sufficient to be observed.
Telomere signal was colocalized with TALT1 in the ALT survivor, suggesting that TALT1 is used as copy template for telomere in the absence of telomerase (Figure 3). Telomere signal of ALT survivors increased compared to that of parental trt-1(ok410) mutant, indicating that telomere is robustly maintained in ALT survivors without telomerase (Figure 4). The signal of PNA probe was greater than that of digoxigenin-labeled probe (Figure 4). Designing probes with PNA oligomer might result in stronger signal than digoxigenin-labeling.
Figure 1: Overview of FISH Experiment. Eggs are harvested by bleaching adult worms and fixed in 2% PFA on a polylysine-coated slide. Samples are freeze-cracked and permeabilized with methanol and acetone for probe penetration. Probes are added to the sample and hybridized overnight at 37 °C. Digoxigenin-labeled probe is detected by immunofluorescence. The samples are imaged and then quantified by the image analysis software. Please click here to view a larger version of this figure.
Figure 2: Telomere FISH in the Dissected Gonads. (A) Telomere (red) was detected by cy3-PNA-(TTAGGC)3 in the distal tip of gonads (arrowhead). The intensity of ALT survivor is greater than that of trt-1(ok410). Z-stack image was rendered with maximum projection. Nuclei indicated by white arrow is blown-up on the upper right corner. Scale bar, 10 µm. (B) Number of telomere spots per nucleus in pachytene stage was measured by visual inspection. N = 50. Error bars, SEM. Please click here to view a larger version of this figure.
Figure 3: Telomere and TALT1 FISH in the Embryos. A representative image of telomere (red) and TALT1 (green) FISH. Telomere and TALT1 probe were hybridized to embryos simultaneously. DNA was counterstained with DAPI (blue). Scale bar, 10 µm. Please click here to view a larger version of this figure.
Figure 4: Quantification of FISH Data. Telomere and TALT1 intensity from Figure 3 were quantified in the image analysis software (a gift from Dr. Peter Lansdorp). Each spot was quantified with threshold level over 15 to exclude non-specific background. T-test was used for evaluating statistical significance. (*p < 0.001). Error bars, SEM. Please click here to view a larger version of this figure.
The main advantage of our protocol is the simplicity of the procedure without noticeable damage to the morphology of cellular structure. Several steps were optimized for C. elegans FISH in this protocol. The critical steps for successful FISH include labeling of probes, fixation of embryos and penetration. Digoxigenin-dUTP labeling method provides an easy-to-use labeling method by PCR or nick-translation. To label long target sequence, nick-translation is preferred. In this case, the probes should be digested with appropriate restriction enzyme to facilitate the penetration of probes. Biotin-dUTP tag is not recommended because biotin-labeled probes produced excess amount of background signal from the cytoplasm. Although endogenous biotin blocking reagent is commercially available, it was not attempted.
This protocol uses isolated embryos to increase the density of cells for efficient quantification. Intestinal nuclei of C. elegans are large in size and are polyploid, which contribute excessive number and intensity of telomere spots compared to the rest of somatic nuclei. For this reason, whole worm is not suitable for quantification of telomeres in C. elegans. In contrast, embryos are appropriate for evaluation of telomere length as they provide homogeneous cells without effect of polyploidy.
This protocol uses 2% PFA fixative that worked fine for telomere FISH. Although glutaraldehyde is reported to result in a lower background signal and harder fixation in RNA in situ hybridization, glutaraldehyde increased autofluorescence significantly18. This excess background was not abolished after treatment of sodium borohydride, which reduces unreacted aldehyde group. For this reason, glutaraldehyde was not used. If the signal-to-noise ratio is low, time of prehybridization can be extended up to several hours to block non-specific binding sites. In addition, time of stringent washing can be increased to decrease background level.
Staining technique in C. elegans can be a challenge for proper permeabilization treatment. C. elegans contains thick cuticular exoskeleton which inhibits penetration of antibodies and probes. Traditional antibody staining method involves the treatment of collagenase, which requires much time and optimization process. The enzymatic penetration method also damages the morphology of the worms in exchange of penetration efficiency. Freeze-crack and methanol-acetone treatment was used to facilitate probe penetration. Freeze-crack is simple and rapid compared to chitinase or yatalase treatment19. Dehydration or rehydration of methanol series did not seem to affect the quality of FISH. These steps were simplified in this protocol. Although it was reported that proteinase K digestion was required for RNA in situ hybridization19, obvious difference was not observed between telomere FISH results with and without proteinase K treatment. In addition, freeze-cracking of embryos provided an easy-to-use method for visualizing many cells simultaneously on a single focal plane for large quantitative analysis.
By using PNA probe, telomere signal was significantly increased compared with that obtained with a DNA probe. This might be due to higher binding affinity for its complementary target and its smaller size (3 repeat compared to 4 repeat). Fluorescently labeled PNA probe also directly binds to its target, minimizing subsequent steps. Strong affinity maintained in the following immunofluorescence steps makes the post-fixation unnecessary, which can avoid background noise.
However, some limitations exist in this technique. One is that the permeabilization step slightly damages the morphology at the cost of efficient probe penetration. Treatment of gonads and embryos with methanol and acetone distorted circularity of nuclei compared to untreated control. For experiments that require perfectly preserved morphology, different permeabilization method should be attempted. Another is that the telomere signal is quantified in arbitrary units. This is mainly due to variations among independent experiments. Ribosomal DNA may be considered as an internal control to normalize each sample. More useful methods can be found in reference 20.
A simple telomere FISH protocol is described here, which requires minimum steps. Many steps are reduced for analysis of large amount of embryos. However, further modification can be made for stronger signal, such as chitinase treatment for penetration, and other innovative trials. In combination with cell culture method, super-resolution imaging may be possible. This protocol may help to discover the novel telomere maintenance mechanism in C. elegans.
The authors have nothing to disclose.
Mutant worm strains were kindly provided by the Caenorhabditis Genetics Center. This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C1277).
PNA probe | PANAGENE | custom order | |
Anti-Digoxigenin-Fluorescein, Fab fragments | Roche | 11207741910 | use 1:200 diluted in PBST |
Digoxigenin-dUTP | Roche | 11573152910 | |
Bovine serum albumin | SIGMA-ALDRICH | A-7906 | |
Paraformaldehyde | SIGMA-ALDRICH | P-6148 | prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS. |
Vectashield | Vector Laboratories | H-1200 | |
Hybridizaiton solution | 3X SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 ug/ml heparin, 100 ug/ml yeast tRNA , 100ug/ml sonicated salmon sperm DNA | ||
Hybridizaiton wash solution | 2X SSC, 50% formamide | ||
Formamide | BIONEER | C-9012 | toxic |
Methanol | Carlo Erba | ||
Acetone | Carlo Erba | ||
Heparin | SIGMA-ALDRICH | H3393 | make 10 mg/ml for stock solution |
Dextran sulfate | SIGMA-ALDRICH | 67578 | |
10X PBS | For 1 Liter DW : 80 g NaCl, 2.0 g KCl, 27 g Na2HPO4:7H2O, 2.4 g KH2PO | ||
PBST | 1X PBS, 0.1% tween-20 | ||
Polysorbate 20 | SIGMA-ALDRICH | P-2287 | Commercial name is Tween-20 |
Poly-L-Lysine solution (0.1 % w/v) | SIGMA-ALDRICH | P-8920 | prepare fresh 0.01 % w/v solution before use |
M9 | 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L | ||
Bleaching solution | 20% sodium hypochlorite, 0.5 M KOH | ||
Antibody buffer | 1X PBST, 1mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic) | ||
Blocking solution | Antibody buffer with 5% bovine serum albumin (BSA) | ||
illustra Microspin G-50 | GE healthcare | 27-53310-01 | |
20X SSC | To make 1L, 175.3 g of NaCl, 88.2 g of sodium citrate, H2O to 1 L, adjust pH to 7.0 | ||
2X SSCT | 2X SSC, 0.1 % tween-20 | ||
10x digoxigenin-dUTP mix | 1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP | ||
PCR purification columns | Cosmo genetech | CMR0112 | |
Glass cleaner / ULTRA CLEAN | Dukssan pure chemicals | 8AV721 | |
Multi-well glass slide | MP biomedicals | 96041205 | |
Nematode growth media | to make 1 L, 3 g of NaCl, 17 g of agar, 2.5 g of peptone, H2O to 974 mL. Autoclave and cool the flask. Add 1 mL of 1M CaCl2, 1 ml of 4 mg/mL cholesterol in ethanol, 1 ml of 1 M MgSO4, 25 mL of 1 M KPO4. | ||
Levamisole | SIGMA-ALDRICH | 196142 | |
Razor | Feather | blade No. 11 | |
Rnase A | Enzynomics | ||
BSA | SIGMA-ALDRICH | A7906 | |
Equipments | |||
Confocal microsope | Zeiss | LSM 510 | EC Plan-Neofluar 100x was used as objective lens. |
Dry block / aluminum block | Labtech | LBH-T03 | Set temperature to 80℃ |
Humid chamber | Plastic box filled with paper towel soaked in DW | ||
Image Analysis Software | Dr. Peter Landsdorp | TFL-telo | http://www.flintbox.com/public/project/502 |